Adoptive immunotherapy has recently produced encouraging clinical results against some forms of cancer. See articles in the Wall Street Journal, Apr. 9, 1987, and Time Magazine, Apr. 20, 1987. The therapy involves removing peripheral blood from a patient, removing red blood cells (RBC's) from the blood to produce a lymphocyte-containing white blood cell (WBC) fraction, incubating the blood fraction in culture medium with interleukin-2 (IL-2) to produce activated, tumor-destroying lymphocytes called LAK cells, and injecting the LAK cells and additional IL-2 into the patient. In some cases IL-2 is injected into the patient before removal of the blood in order to stimulate production of lymphocytes.
One objection to adoptive immunotherapy is that it is very expensive. One reason it is expensive is that the current procedure for producing LAK cells is labor-intensive and time consuming. This procedure is described in Muul et al., "Large scale production of human lymphokine activated killer cells for use in adoptive immunotherapy," Journal of Immunological Methods, 88: 265-275 (1986). As described in Muul et al., in order to generate enough LAK cells for a single treatment about 2.times.10" lymphocytes were obtained by 10 successive leukaphereses of peripheral blood. In each leukapheresis, about 10-12 liters of whole blood were processed in an automated cell separator over a 4-hour period to produce a 400-500 ml leucocyte fraction. This fraction was diluted with 2 parts of a salt solution, then poured into 50 ml conical centrifuge tubes (40 ml/tube, approx. 30-40 tubes) and underlayed with 10 ml Ficoll-Hypaque solution. The contents were centrifuged, causing separation into a platelet-rich supernatant layer, a lymphocyte-rich layer, a Ficoll-Hypaque layer, a granulocyte layer and an RBC layer. The supernatant was removed from each tube and discarded. The lymphocyte-rich fraction floating on the Ficoll-Hypaque was removed from each tube; these fractions were pooled and washed three times by suspension in salt solution and centrifugation. Since these steps must be repeated for each leukapheresis, 300-400 tubes must be handled for a single treatment.
Haemonetics Corporation of Braintree, Mass., markets an automated blood cell separator known as the Haemonetics V-50, which utilizes a 2-port conically-shaped centrifuge bowl similar to the bowl described in U.S. Pat. No. 3,145,713. The V-50 can be operated according to a standard leukapheresis protocol or according to a Surge.RTM. lymphocytopheresis protocol. The latter procedure, as described in U.S. Pat. Nos. 4,464,167 and 4,416,654, involves intermittent elutriation with previously-separated plasma, and is capable of providing more precise fractions of platelets, WBC's and RBC's than can be achieved with standard leukapheresis; it is referred to hereinafter elutriation leukapheresis.
For LAK cell processing, Haemonetics recommends use of the V-50 to separate a Buffy coat composed mostly of platelets and WBC's, followed by a secondary separation using Ficoll-Hypaque to provide a density gradient in the same centrifuge bowl for isolation of mononuclear cells (lymphocytes and monocytes) from the Buffy coat. Although this procedure is much less time-consuming and labor-intensive than the standard ficoll centrifugation described in Muul et al., it would be desirable to eliminate the ficoll separation step because it adds to the cost and can cause a reduced yield of lymphocytes. However, up to now it has been considered essential by those skilled in the art to conduct a ficoll separation in order to obtain a lymphocyte fraction sufficiently free of RBC's and granulocytes to be useful for production of LAK cells. It was assumed that RBC's and granulocytes would unduly interfere with the activation of the lymphocytes.